This application is to renew a grant to study RNA synthesis in minus-strand RNA viruses. Nonsegmented negative-sense (NNS) RNA viruses include some of the most significant human pathogens that are an ongoing threat to US public health. For measles, mumps and rabies there are licensed vaccines, but for most NNS RNA viruses there are no vaccines and no antiviral drugs. Our long-term objective is to understand the mechanisms by which the replication machinery of vesicular stomatitis virus (VSV), a prototype of the NNS RNA viruses, functions. VSV is the ideal choice for such studies because it is the only NNS RNA virus for which robust transcription can be reconstituted in vitro from purified recombinant components. The catalytic core of the RNA synthesis machinery is a 241 kDa large protein (L) that contains an RNA dependent RNA polymerase (RdRP), a polyribonucleotidyltransferase (PRNTase) that caps the mRNA, and a dual specificity mRNA cap methyltransferase (MTase). During mRNA synthesis, those activities are coordinated so that the nascent mRNA is capped, methylated and polyadenylated. Although L contains all the enzymatic activities for RNA synthesis, it requires a 29 kDa phosphoprotein (P) that bridges interactions between L and the nucleocapsid protein (N) that completely coats the RNA template. In the last grant period, we obtained the first structural insights into the polymerase complex of an NNS RNA virus and developed unique tools and reagents that will permit us to obtain an atomic-level model of the VSV polymerase complex. Our underlying hypothesis is that the catalytic activities of L in RNA polymerization, mRNA cap addition and cap methylation which reside within structurally separate domains are coordinated by the presence of the P and the template associated N to regulate their activities during mRNA synthesis, and to downregulate them during assembly by complex formation with the viral matrix protein (M). A major gap in understanding the machinery of RNA synthesis is the current resolution of structures of L. We have in hand an interpretable density map of a VSV L-P complex from cryo EM. During the next funding period, we will continue to use cryo electron microscopy (EM), negative-stain EM, X-ray crystallography, in vitro biochemistry of polymerase and molecular virology to provide unique structural and functional insights into the VSV polymerase during distinct stages of RNA synthesis and assembly. We will: (i) determine a complete molecular model of the VSV polymerase complex; (ii) determine how the template associated N protein is displaced from the RNA, and (iii) determine the mechanism by which M protein downregulates polymerase activity. The successful completion of this work will provide an atomic-level structure of an NNS RNA virus polymerase complex and new mechanistic insights into the function and regulation of this RNA synthesis machine during transcription, replication and assembly. Those results may help in the rational attenuation of other related pathogenic human viruses for vaccine purposes, and for the development of antiviral therapeutics.